We are engaged in analysis of the lytic replication of two human herpesviruses: herpes simplex virus (HSV) and Epstein-Barr virus (EBV). We are studying the purified protein products of the viral genes that participate in DNA replication in lytically-infected cells, using both biochemical and molecular genetic approaches to understand the function of these polypeptides in detail. Our recent results regarding HSV proteins can be summarized as follows: 1) UL9, the viral protein that initiates DNA replication, binds to its cognate binding site as a complex of two dimeric molecules; 2) The HSV primase, unlike any other known eukaryotic primases, but similar to several prokaryotic primases, has a requirement for a specific templace sequence; and 3) The EBV BALF2 gene product is a single-stranded DNA binding protein that binds cooperatively to single-stranded DNA. Between 15 and 25 bases of single-stranded DNA are required for efficient binding. In addition, we have the following work in progress. We have constructed a large number of deletion and insertion markers of two of the subunits of the HSV helicase-primase. These mutant polypeptides are being analyzed to provide information regarding the structure of the heterotrimeric helicase primase complex and to determine the biological segnificance of certain biochemical assays that require these proteins. We have also begun to analyse an intertypic recombinant virus (HSV-1 x HSV-2) that shows a profound specific defect in the ability to carry out DNA replication in neuronal cells. Genetic analysis of this virus has shown that the defect can be traced to the helicase gene derived from HSV-2. We have cloned the helicase gene from this virus, and a number of experiments are in progress to determine the underlying mechanism of its neuronal-specific defect.